Highly phosphorylated and sulfated recombinant factor ix

ABSTRACT

The present invention relates to a purified recombinant blood coagulation factor IX (rFIX) preparation, wherein at least 25% of the rFIX in the preparation is fully phosphorylated and sulfated, a cell culture expressing a rFIX resulting in said preparation, a pharmaceutical composition for treating a bleeding disorder comprising said preparation, and a method for treating a bleeding disorder comprising the step of administering said preparation to a patient in need thereof.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Nos. 60/779,830, filed Mar. 7, 2006 and 60/789,857, filedApr. 5, 2006, the disclosures of which are hereby incorporated byreference in their entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to a purified recombinant bloodcoagulation factor IX (rFIX) preparation having at least 25% and lessthan 98% of fully phosphorylated and sulfated rFIX, a cell cultureexpressing a rFIX resulting in said preparation, a pharmaceuticalcomposition for treating a bleeding disorder comprising saidpreparation, and a method for treating a bleeding disorder comprisingthe step of administering said preparation to a patient in need thereof.

BACKGROUND OF THE INVENTION

Hemophilia B, a hereditary recessive bleeding disorder, is successfullytreated by replacement therapy consisting of the administration ofpreparations of human plasma derived (pdFIX) or recombinant coagulationfactor IX (rFIX). The commercially available recombinant product, whichis marketed under the trade name Benefix™, is manufactured by usingstable transfected Chinese hamster ovary (CHO) cells co-expressing rFIXtogether with endopeptidase PACE/Furin, and is highly purified viamultiple filtration and chromatographic steps (Kaufman et a., 1986;Wasley et al., 1993; Harrison et a., 1998). In clinical studies,Benefix™ has been shown to be safe and effective, but a 20 to 50% higherdosage than for pdFIX is needed for successful treatment. This is due toa 30 to 50% lower in vivo recovery for CHO derived rFIX than for pdFIX,as revealed by pharmacokinetic data collected from pre-clinical andclinical studies, where pdFIX and rFIX are compared in different animalmodels (Keith, Jr. et al., 1995; Brinkhous et a., 1996; Schaub et al.,1998; McCarthy et al., 2002), and clinical studies in hemophilia Bpatients (Keith, Jr. et al., 1995; White et al., 1997; White et al.,1998; Bjorkman et al., 2001; Roth et al., 2001; Ewenstein et al., 2002;Poon et al., 2002; Ragni et al., 2002; Kisker et al., 2003; Shapiro etal., 2005a). The circulating half-life of rFIX is not distinguishablefrom pdFIX preparations.

Biochemical comparison between pdFIX and CHO derived rFIX revealeddifferences in post-translational modifications (Bond et al., 1998). Thelower degree of phosphorylation of a unique site at theactivation-peptide amino acid serine 155 and the lower degree ofsulfation of tyrosine 158 have been assigned to the lower in-vivorecovery of rFIX (White et al., 1997; Kaufman, 1998), althoughexperimental evidence to proof this assumption has not been publishedto-date. These two modifications were identified to occur at less than15% for the tyrosine-sulfation and at less than 1% for the serinephosphorylation in the recombinant protein, whereas the plasma derivedprotein has both modifications to more than 90% completed. Similarpharmacokinetic properties to Benefix™ were found formyotube-synthesized rFIX after adeno-associated viral vector mediatedgene delivery in a mouse model (Arruda et al., 2001).

Therefore, a strong need exists for a new rFIX preparation which can beadministered in a lower dosage than conventional rFIX preparation for asuccessful treatment.

Thus, it is an object of the present invention to provide a new rFIXpreparation, wherein the rFIX has an improved in vivo recovery.

SUMMARY OF THE INVENTION

The present invention relates to a purified recombinant bloodcoagulation factor IX (rFIX) preparation having at least 25% and lessthan 98% of fully phosphorylated and sulfated rFIX. The rFIX is forexample expressed in a host cell type with the ability to perform thephosphorylation and sulfation as posttranslational modifications.Additionally, a cell culture expressing rFIX resulting in a purifiedrFIX preparation having at least 25% and less than 98% of fullyphosphorylated and sulfated rFIX, e.g. a HEK293-derived cell culture, isprovided according to the present invention. Further, the presentinvention relates to a pharmaceutical composition comprising a purifiedrFIX preparation having at least 25% and less than 98% of fullyphosphorylated and sulfated rFIX for treating a bleeding disorder, e.g.hemophilia B. It is another object of the present invention to provide amethod for treating a bleeding disorder comprising the step ofadministering a pharmaceutical composition comprising a purified rFIXpreparation having at least 25% and less than 98% of fullyphosphorylated and sulfated rFIX for treating a bleeding disorder.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a purified recombinantblood coagulation factor IX (rFIX) having at least 25% of fullyphosphorylated and sulfated rFIX. The degree of phosphorylation andsulfation may be identified by determining the percentage of fullyphosphorylated and sulfated rFIX by mass spectrometry (MS), e.g.,electrospray-ionization quadrupole time of flight mass spectrometry(ESI-QTOF-MS). When measured with ESI-QTOF-MS, the percentage of fullyphosphorylated and sulfated rFIX in a plasma derived FIX sample used asa standard is about 98%. For conventional rFIX-preparations, e.g.Benefix™, sulfation degree can be less than 15% and phosphorylationdegree less than 1%. Any phosphorylation or sulfation degree leading toan increased in vivo recovery compared to Benefix™ can be determined ashigh, but at least 25% sulfation and phosphorylation and less than 98%phosphorylation and sulfation is encompassed by the present invention.In one example, the range is 30%-95%, 35%-90%, 40%-85%, 40%-75%,40%-65%, 40-60%, 40-55%, or 45-50%. In one example of the presentinvention, the percentage of fully phosphorylated and sulfated rFIXmaterial after purification is at least 28%. In another example of thepresent invention, the percentage of fully phosphorylated and sulfatedrFIX material after purification is at least 35%. In one example of thepresent invention, the percentage of fully phosphorylated and sulfatedrFIX material after purification is at least 46%. In one further exampleof the present invention, the percentage of fully phosphorylated andsulfated rFIX material after purification is at least 49%. In anotherexample of the present invention, the percentage of fully phosphorylatedand sulfated rFIX material after purification is at least 53%.

According to the present invention, the term “recombinant bloodcoagulation factor IX” does not underlie a specific restriction and mayinclude any rFIX, heterologous or naturally occurring, obtained viarecombinant DNA technology, or a biologically active derivative thereof.In certain embodiments, the term encompasses proteins and nucleic acids,e.g., gene, pre-mRNA, mRNA, and polypeptides, polymorphic variants,alleles, mutants, and interspecies homologs that: (1) have an amino acidsequence that has greater than about 60% amino acid sequence identity,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% or greater amino acid sequence identity, over a region of at leastabout 25, 50, 100, 200, 300, 400, 450, or more amino acids (up to thefull length sequence), to a polypeptide encoded by a referenced nucleicacid or an amino acid sequence described herein; (2) specifically bindto antibodies, e.g., polyclonal antibodies, raised against an immunogencomprising a referenced amino acid sequence as described hereinimmunogenic fragments thereof, and conservatively modified variantsthereof, (3) specifically hybridize under stringent hybridizationconditions to a nucleic acid encoding a referenced amino acid sequenceas described herein, and conservatively modified variants thereof, (4)have a nucleic acid sequence that has greater than about 95%, greaterthan about 96%, 97%, 98%, 99%, or higher nucleotide sequence identity,over a region of at least about 25, 50, 100, 150, 200, 250, 500, 1000,2000, 2500 or more nucleotides (up to the full length sequence), to areference nucleic acid sequence as described herein. A polynucleotide orpolypeptide sequence is typically from a mammal including, but notlimited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster;cow, pig, horse, sheep, or any mammal. The nucleic acids and proteins ofthe invention are recombinant molecules (e.g., heterologous and encodingthe wild type sequence or a variant thereof, or non-naturallyoccurring). Reference polynucleotide and polypeptide sequences include,e.g., Accession Nos. NM_000133; BC109214; BC109215; J00137P M11309; andJ00136 (see, e.g., Mammalian Gene Collection Program Team, PNAS USA99:16899-16903 (2002); Autin et al., J. Thromb. Haemost. 3:2044-2056(2005); Jaye et al., NAR 11:2325-2335 (1983); McGraw et al., PNAS82:2847-2851 (1985) Choo et al., Nature 299:178-180 (1982); and Kurachiand Davie, PNAS 79:6461-6464 (1982)). In one embodiment, there are 2point mutations in the Factor IX nucleic acid sequence as compared toNM_(—)000133: position 57: CTT->CTC Leu->Leu (no amino acid change); andposition 580: ACT->GCT Thr->Ala (amino acid change).

The term “fully phosphorylated and sulfated rFIX refers to the degree ofphosphorylation and sulfation as identified by determining thepercentage of fully phosphorylated and sulfated rFIX by massspectrometry (MS), e.g., electrospray-ionization quadrupole time offlight mass spectrometry (ESI-QTOF-MS). When measured with ESI-QTOF-MS,the percentage of fully phosphorylated and sulfated rFIX in a plasmaderived FIX sample used as a standard is about 98%.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous or non-naturally occurring nucleic acid or protein, or thealteration of a native nucleic acid or protein, or that the cell isderived from a cell so modified. Thus, for example, recombinant cellsexpress genes that are not found within the native (non-recombinant)form of the cell, or express wild type and variant genes that are not inthe native position in the genome of the cell, or express native genesthat are otherwise abnormally expressed, under expressed or notexpressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. In one example, this term refers to a nucleic acid that isnot in its native position in the genome. In another example, thenucleic acid is recombinantly produced, having two or more sequencesfrom unrelated genes arranged to make a new functional nucleic acid,e.g., a promoter from one source and a coding region from anothersource. Similarly, a heterologous protein indicates that the proteincomprises two or more subsequences that are not found in the samerelationship to each other in nature (e.g., a fusion protein), or thatit is a protein derived from a heterologous nucleic acid.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength pH. The Tm is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, or 10 times background hybridization.Exemplary stringent hybridization conditions can be as following: 50%formamide, 5× SSC, and 1% SDS, incubating at 42° C., or, 5× SSC, 1% SDS,incubating at 65° C., with wash in 0.2× SSC, and 0.1% SDS at 65° C.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specifiedregion, when compared and aligned for maximum correspondence over acomparison window or designated region) as measured using a BLAST orBLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like).Such sequences are then said to be “substantially identical.” Thisdefinition also refers to, or may be applied to, the complement of atest sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions. Asdescribed below, the algorithms can account for gaps and the like.Identity exists over a region that is at least about 25 amino acids ornucleotides in length, or over a region that is 50-100 amino acids ornucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

An example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always>0) and N (penalty score for mismatchingresidues; always<0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff& Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

As used herein, the term “biologically active derivative” includes anyderivative of a protein, protein complex or polypeptide havingsubstantially the same functional and/or biological properties of rFIXsuch as binding properties, and/or the same structural basis, such as apeptidic backbone. Minor deletions, additions and/or substitutions ofamino acids of the polypeptide sequence of rFIX which are not alteringthe biological activity of said polypeptide are also included in thepresent application as biologically active derivatives.

The rFIX according to the present invention may be derived from anyvertebrate, e.g. a mammal. In one specific example of the presentinvention, the rFIX is human FIX.

The rFIX according to the present invention may be produced by anymethod known in the art. This may include any method known in the artfor the production of recombinant DNA by genetic engineering, e.g. viareverse transcription of RNA and/or amplification of DNA. Additionally,the recombinant DNA coding for rFIX, e.g. a plasmid, may also contain aDNA sequence encoding a selectable marker for selecting the cells whichhave been successfully transfected with the plasmid. In an example ofthe present invention, the plasmid may also confer resistance to aselectable marker, e.g. to the antibiotic drug G418, by delivering aresistance gene, e.g. the neo resistance gene conferring resistance toG418.

The production of rFIX may include any method known in the art for theintroduction of recombinant DNA into eukaryotic cells by transfection,e.g. via electroporation or microinjection. For example, the recombinantexpression of human FIX can be achieved by introducing an expressionplasmid containing the human FIX encoding DNA sequence under the controlof one or more regulating sequences such as a strong promoter, into asuitable host cell line by an appropriate transfection method resultingin cells having the introduced sequences stably integrated into thegenome. The calcium-phosphate co-precipitation method is an example of atransfection method which may be used according to the presentinvention.

The production of rFIX may also include any method known in the art forthe cultivation of said transformed cells, e.g. in a continuous orbatchwise manner, and the expression of the rFIX, e.g. constitutive orupon induction. In one specific example of the present invention thenucleic acid coding for rFIX contained in the host organism of thepresent invention is expressed via an expression mode selected from thegroup consisting of induced, transient, and permanent expression. Anyexpression system known in the art or commercially available can beemployed for the expression of a recombinant nucleic acid encoding rFIX,including the use of regulatory systems such as suitable, e.g.controllable, promoters, enhancers etc.

The production of rFIX may also include any method known in the art forthe isolation of the protein, e.g. from the culture medium or byharvesting the transformed cells. For example, the rFIX-producing cellscan be identified by isolating single-cell derived populations i.e. cellclones, via dilution after transfection and optionally via addition of aselective drug to the medium. After isolation the identified cell clonesmay be cultivated until confluency in order to enable the measurement ofthe rFIX content of the cell culture supernatant by enzyme-linkedimmuno-sorbent assay (ELISA) technique. Additionally, rFIX secreted bythe cells may be identified for example by growing the cells in theabsence of any growth promoting fetal bovine serum or componentsthereof. Vitamin K is added at appropriate concentrations to improve thefunctional properties of the rFIX protein. In one specific example ofthe present invention, the supernatant is harvested 24 hours aftertransfection. After identification, high rFIX producing cell clones mayfor example be further propagated and/or stored via cryopreservation. Inone example of the present invention, the rFIX is co-expressed withvitamin K reductase complex subunit 1 (VKORC 1) and/or furin.

Additionally, the production of rFIX may include any method known in theart for the purification of rFIX, e.g. via anion exchange chromatographyor affinity chromatography. In one embodiment rFIX can be purified fromcell culture supernatants by semi-affinity calcium-dependent anionexchange chromatography, e.g. in an endotoxin-free system. The purifiedrFIX may be analyzed by methods known in the art for analyzingrecombinant proteins, e.g. the ELISA technique. In addition, the proteinintegrity and activity may be assessed by measuring activated partialthromboplastin time (APTT) and by electrophoresis techniques includingimmuno-blotting.

Examples for the detection systems of the phosphorylation and sulfationof rFIX are known to a person skilled in the art. For example, degreesof phosphorylation and sulfation can be analyzed by LC-MS. It is withinthe knowledge of a person skilled in the art to choose the optimalparameters, such as buffer system, temperature and pH for the respectivedetection system to be used.

In one specific example of the present invention, the rFIX according tothe present invention is expressed in a host cell type with the abilityto perform the phosphorylation and sulfation as posttranslationalmodifications. The ability to phosphorylate and sulfate correspondingresidues on rFIX of rFIX expressing host cell lines may be for exampleanalyzed by mass-spectrometric analysis of the rFIX derived from thesecell lines. For example, cell clones exhibiting the ability to addphosphorus-containing groups and sulfur-containing groups to thesynthesized rFIX molecules, may be identified by determining thepercentage of fully phosphorylated and sulfated rFIX by massspectrometry (MS) after chromatographic purification of rFIX from cellculture supernatants. This may be accomplished, by e.g. binding rFIXprotein to an anion exchange column and eluting fully carboxylated rFIXvia the addition of Ca(II) ions. The conversion of collected rFIXpreparations into peptides may be achieved, e.g. by tryptic digestion,optionally followed by an enzymatical removal of glycosidic residues. Inthe following, the peptides may be separated, e.g. by reversed phaseHPLC, and analyzed by electro-spray-ionization quadrupole time of flightmass spectrometry (ESI-QTOF-MS). The degree ofphosphorylated/non-phosphorylated and/or sulfated/non-sulfated peptidemay be estimated by quantification of corresponding signals.

The host cell type according to the present invention may be anymammalian cell with the ability to perform the phosphorylation andsulfation as posttranslational modifications of rFIX. For example saidmammalian cell is derived from a mammalian cell line, like for example acell line selected from the group consisting of SkHep-, CHO-, HEK293-,and BHK-cells. In a specific example of the present invention, the rFIXis expressed in HEK293-derived cells.

The improved pharmacokinetic properties of higher phosphorylated andsulfated rFIX molecules may be confirmed e.g. in FIX-knockout mousemodels.

According to the present invention, the in vivo recovery of rFIX afterinjection into an individuum is the observed peak plasma concentrationrelative to the expected peak concentration based on body weight orplasma volume. It is calculated from the maximum rFIX concentration risefrom baseline and is expressed as U/dL or μg/dL increase per dose (U/kgor μg/kg) injected based on bodyweight. If based on plasma volume, thein-vivo recovery can be calculated as percentage of U found per U dosed(Shapiro et al., 2005b).

A further aspect of the present invention is the provision of a cellculture comprising cells expressing a rFIX resulting in a purified rFIXpreparation having at least 25% of fully phosphorylated and sulfatedrFIX.

The cell culture of the present invention may comprise any cell culturewhich contains cells capable of expressing rFIX and performing thephosphorylation and sulfation as posttranslational modifications ofrFIX. Examples of suitable cells are listed above. In one specificexample, the cell culture of the present invention is an eukaryotic cellculture characterized by producing one or more pharmacologically activerFIX having an improved in vivo recovery.

In one example of the present invention the cell culture of the presentinvention comprises a host organism as defined above.

There is no particular limitation to the media, reagents and conditionsused for culturing the cells in the cell culture of the presentinvention including culturing the cells in a continuous or batchwisemanner. In one example of the present invention the cells are culturedunder serum-free or serum- and protein-free conditions. In a furtherexample of the present invention conditions are employed under whichcells which contain a recombinant nucleic acid coding for rFIX areselectively proliferated, e.g. by using a selective medium.

The desired rFIX protein which has been expressed by the cells of theselected host organism and which, dependent on thetransfection/vector-system used, is contained in the cells or secretedinto the medium for culturing cells, can be isolated/recovered from thecell culture using methods known in the art, as mentioned herein before.

In one specific example of the present invention, the cell cultureaccording to the present invention comprises HEK293-derived cells. Inanother example of the present invention, the cells are switched toserum-free medium at confluency. Further, in another example of thepresent invention, the supernatants are harvested every day for at leasttwo weeks.

The cell culture according to the present invention produces for examplea rFIX resulting in a purified rFIX preparation having afterpurification at least 25% of fully phosphorylated and sulfated rFIX. Insome embodiments, the cell culture produces about 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550,1600, 1650, 1700, 1750, 1800, 1850, 1900, 2000, 2050, 2100, 2150, 2200,2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800,2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400,3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 3950, 4000,4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600,4650, 4700, 4750, 4800, 4850, 4900, 4950 or about 5000 mU FIX per 10⁶cells per day.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising the above defined purified rFIX preparationhaving at least 25% of fully phosphorylated and sulfated rFIX fortreating a bleeding disorder, e.g. a bleeding disorder associated withfunctional defects of FIX or deficiencies of FIX.

The expression “bleeding disorder associated with functional defects ofFIX or deficiencies of FIX” as used herein includes bleeding disorders,wherein the cause of the bleeding disorder may be selected from thegroup consisting of a shortened in vivo-half-life of FIX, alteredbinding properties of FIX, genetic defects of FIX, and a reduced plasmaconcentration of FIX. Genetic defects of FIX comprise for exampledeletions, additions and/or substitution of bases in the nucleotidesequence encoding FIX whose absence, presence and/or substitution,respectively, has a negative impact on the activity of FIX. In oneexample of the present invention, the bleeding disorder is hemophilia B.Symptoms of such bleeding disorders include, e.g., severe epistaxis,oral mucosal bleeding, hemarthrosis, hematoma, persistent hematuria,gastrointestinal bleeding, retroperitoneal bleeding,tongue/retropharyngeal bleeding, intracranial bleeding,trauma-associated bleeding.

According to the methods of the present invention, a compositioncomprising the rFIX is administered by any parenteral (e.g.,intravenously, intramuscularly, subcutaneously, or intraperitoneally) ornon-parenteral route (e.g., orally). Pharmaceutical compositionscomprising the rFIX described herein may also contain suitableexcipients (e.g., carriers, buffers, and the like). Any suitable carrierknown to those of ordinary skill in the art may be employed in thepharmaceutical compositions of this invention. Suitable carriersinclude, for example, water, saline, alcohol, a fat, a wax, a buffer, asolid carrier, such as mannitol, lactose, starch, magnesium stearate,sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesiumcarbonate. Suitable buffers include, e.g., e.g., neutral buffered salineor phosphate buffered saline. Additional suitable excipients include,e.g., carbohydrates (e.g., glucose, mannose, sucrose or dextrans),mannitol, proteins, polypeptides or amino acids such as histindine orglycine, antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, detergents (e.g., fatty acid esters of sorbitanpolyethoxylates such as, for example, polysorbate 20, polysorbate 60, orpolysorbate 80), solutes that render the formulation isotonic, hypotonicor weakly hypertonic with the blood of a recipient, suspending agents,thickening agents and/or preservatives. Alternatively, compositions ofthe present invention may be formulated as a lyophilizate.

The compositions described herein may be administered as part of asustained release formulation (i.e., a formulation such as a capsule orsponge that effects a slow release of compound followingadministration). Such formulations may generally be prepared using wellknown technology (see, e.g., Coombes et al. (1996) Vaccine14:1429-1438). Sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.

Carriers for use within such formulations are biocompatible, and mayalso be biodegradable; preferably the formulation provides a relativelyconstant level of active component release. Such carriers includemicroparticles of poly(lactide-co-glycolide), as well as polyacrylate,latex, starch, cellulose and dextran. Other delayed-release carriersinclude supramolecular biovectors, which comprise a non-liquidhydrophilic core (e.g., a cross-linked polysaccharide oroligosaccharide) and, optionally, an external layer comprising anamphiphilic compound (see, e.g., WO 94/20078; WO 94/23701; and WO96/06638). The amount of active compound contained within a sustainedrelease formulation depends upon the site of implantation, the rate andexpected duration of release and the nature of the condition to betreated or prevented.

The pharmaceutical compositions may be presented in unit-dose ormulti-dose containers, such as sealed ampoules or vials. Such containersare preferably hermetically sealed to preserve sterility of theformulation until use. In general, formulations may be stored assuspensions, solutions or emulsions in oily or aqueous vehicles.Alternatively, a pharmaceutical composition may be stored in afreeze-dried condition requiring only the addition of a sterile liquidcarrier immediately prior to use.

The present invention further relates to the use of a purified rFIXpreparation having at least 25% of fully phosphorylated and sulfatedrFIX in the manufacture of a medicament for treating a bleedingdisorder.

One aspect of the present invention involves using the rFIX compositionsdescribed herein to treat, prevent or alleviate symptoms of the bleedingdisorders associated with functional defects of FIX or deficiencies ofFIX such as, for example, Hepatitis B. As used herein, a “subject” or a“patient” refers to any warm-blooded animal, such as, for example, aprimate, preferably a human.

A “therapeutic dose” or “therapeutically effective amount” or “effectiveamount” of rFIX or a composition comprising rFIX is an amount of therFIX or composition comprising rFIX which prevents, alleviates, abates,or reduces the severity of symptoms of bleeding disorders associatedwith functional defects of FIX or deficiencies of FIX.

Frequency of administration of the rFIX compositions described herein,as well as dosage, will vary from individual to individual, and may bereadily established using standard techniques. Often 1, 2, 3, 4, or 5doses are administered each week. In some cases, the doses areadministered daily. In some cases, doses are administered 1, 2, 3, 4, ormore times per day. Doses can also be administered on demand (e.g.,following a trauma that causes bleeding in a subject or prior to ascheduled medical procedure expected to cause bleeding in a subject suchas, for example surgery or dental work). A therapeutic dose is an amountof a compound that, when administered as described above, is capable ofpromoting an increased in vivo recovery of rFIX (e.g., as measured usingthe methods set forth in Example 3 below) or Factor IX activity (e.g.,as measured by APTT assays as set forth in, e.g., Chen et al, Adv Ther.2003 September-October; 20(5):231-6) following administration of thecompositions to the individual. The compositions should also be capableof causing a response that leads to an improved clinical outcome (e.g.,improved clotting time) in patients receiving the rFIX as compared topatients who do not receive such treatment. Such responses may generallybe evaluated using samples obtained from a patient before and aftertreatment. Suitable dose sizes will vary with the body weight of thepatient, type of hemorrhage to be treated or prevented, and the desiredplasma FIX concentration, but will typically range from about 10-150,20-100, 20-5-, or 40-50 International Units (IU) per kg body weight. AnIU of FIX activity per kg body weight is typically equal to the FIXactivity in 1 ml of fresh plasma and increases the FIX plasmaconcentration by 1%.

It is another object of the present invention to provide a method fortreating a bleeding disorder comprising the step of administering apharmaceutical composition comprising a purified rFIX preparation havingat least 25% of fully phosphorylated and sulfated rFIX, e.g. fortreating a bleeding disorder, e.g. to a patient in need thereof. In oneexample of the method for treating a bleeding disorder according to thepresent invention, the purified rFIX preparation having at least 25% offully phosphorylated and sulfated rFIX, the pharmaceutical compositionand the bleeding disorder are selected from the group consisting of thepurified rFIX preparation as defined above, the pharmaceuticalcomposition as defined above and the bleeding disorder as defined above.

The present invention will be further illustrated in the followingexamples, without any limitation thereto.

EXAMPLES Example 1 Pharmacokinetics of De-Phosphorylated pdFIX inComparison with pdFIX and rFIX-Product Benefix™

In order to trace altered in vivo recovery properties of FIX back tophosphorylation, the following study is performed: A pdFIX preparationis enzymatically de-phosphorylated using λ-phosphatase in order tocompare the pharmacokinetics of the pdFIX molecule obtained after thisprocedure with Benefix™ and non-de-phosphorylated pdFIX in aFIX-knockout mouse model.

Briefly, pdFIX is de-phosphorylated with λ-phosphatase and purified viaanion exchange chromatography. Benefix™, de-phosphorylated pdFIX, andpdFIX are formulated in the same buffer and administered intravenously(i.v.) to FIX-knockout mice at a dosage of 200 μg/kg and a volume of 10ml/kg. Citrated plasma samples are taken after 15 min, 30 min, 1, 2, 4,and 16 hours. Each treatment is carried out with 10 animals pertreatment and time point. FIX concentrations and activities aredetermined via ELISA and APTT. In vivo recovery is calculated from thehighest FIX concentration value found within the first hour and isexpressed as percentage of the administered dose. Half-life iscalculated using a one-phase least square linear regression model oflogarithmic transformed ELISA or APTT values. For each treatment themedian values and the 95% confidence intervals of APTT and ELISAmeasurements are calculated. The results are shown in Table I for invivo recovery and in Table II for half-life. Concerning recovery, thedifferences of pair wise comparisons of median values from bothmeasurement methods are significant for Benefix™ in comparison to pdFIXas well as for de-phosphorylated pdFIX to pdFIX, but not significantbetween Benefix™ and de-phosphorylated pdFIX. Half-life is found to beslightly higher for the rFIX product Benefix™ than for pdFIX andde-phosphorylated pdFIX.

Similar in vivo recoveries of de-phosphorylated pdFIX and rFIX arefound, which are both 40 to 60% lower than the observed in vivo recoveryof pdFIX. It can be concluded from this study, that at least theenzymatic removal of the phosphate group at serine 158 from the FIXactivation peptide, which is the only phosphorylation site within theFIX protein, converts pdFIX into a species with an in vivo recoverycomparable to CHO-derived rFIX. Therefore, phosphorylation andeventually sulfation are valid targets when aiming at the development ofcell lines for the production of an improved rFIX product.

TABLE I In vivo recovery median values (%) of rFIX product Benefix ™,pdFIX, and enzymatically de-phosphorylated pdFIX found in FIX-knockoutmice and as determined by ELISA and APTT clotting assay measurements. %in vivo recovery % in vivo recovery based on ELISA based on APTT 95% 95%Median confidence Median confidence Treatment values interval valuesinterval Benefix ™ 7.2 5.4 to 9.3 6.0 5.1 to 9.0 Enzymatically 9.5 8.1to 10.8 8.4 6.9 to 11.3 dephosphorylated pdFIX pdFIX 17.0 13.1 to 20.015.0 13.6 to 20.6

TABLE II Half-life median values (hours) of rFIX product Benefix ™,pdFIX, and enzymatically de-phosphorylated pdFIX found in FIX-knockoutmice and as determined by ELISA and APTT clotting assay measurements.Half life (hours) Half-life (hours) based on ELISA based on APTT 95% 95%Median confidence Median confidence Treatment values interval valuesinterval Benefix ™ 6.5 5.9 to 7.2 8.9 7.9 to 12.0 Enzymatically 6.1 5.7to 6.7 6.7 6.3 to 7.4 de-phosphorylated pdFIX pdFIX 5.1 4.5 to 5.8 6.66.0 to 7.6

Example 2 Recombinant Expression of FIX in Cell Culture and Screeningfor Cell Lines Exhibiting a High Degree of rFIX-Phosphorylation andSulfation

The recombinant expression of human FIX is achieved by introducing anexpression plasmid containing the human FIX encoding DNA sequence underthe control of a strong promoter into the host cell line by anappropriate transfection method resulting in cells having the introducedsequences stably integrated into the genome. The transfection methodused is a so-called calcium-phosphate co-precipitation method. Theplasmid also confers resistance to a selectable marker antibiotic drugG418 by delivering the neo resistance gene.

For the identification of rFIX-producing cells, after transfection andaddition of the selective drug to the medium, the cell suspension isdiluted to enable isolation of single-cell derived populations, i.e.cell clones. After isolation, these cell clones are cultivated untilconfluency to enable the measurement of the rFIX content of the cellculture supernatant by ELISA technique. For that purpose, the cells aregrown in the absence of any growth promoting fetal bovine serum orcomponents thereof to ensure the identification of the cells secretingrFIX. To ensure a fully functional rFIX protein, vitamin K is added atappropriate concentrations. The supernatant is harvested after 24 hoursand can be analyzed by ELISA technique. In addition, the proteinintegrity and activity is assessed by measuring APTT and byelectrophoresis techniques including immuno-blotting. High rFIXproducing cell clones are further propagated and stored viacryopreservation.

To identify cell clones exhibiting the ability to add phosphor andsulfate groups to the synthesized rFIX molecules, the percentage offully phosphorylated and sulfated rFIX is determined by massspectrometry (MS) after chromatographic purification of rFIX from cellculture supernatants. This is accomplished by binding rFIX protein to ananion exchange column and eluting fully carboxylated rFIX via theaddition of Ca(II) ions as described in EP 0669342. Thus, collected rFIXpreparations are converted to peptides by tryptic digestion, andglycosidic residues are removed enzymatically. These peptides areseparated by reversed phase HPLC and are analyzed by ESI-QTOF-MS. Thedegree of phosphorylated and sulfated peptide is estimated byquantification of corresponding signals.

These techniques are used to generate cell lines producing rFIX. In thisstudy, the recombinant expression of FIX is compared in 4 different hostcell types (CHO, SkHep, BHK, HEK293) after stable transfection, andscreening of appropriate producer cell lines. The percentages ofphosphorylated and sulfated rFIX protein isoform from total rFIX afterdown-stream purification are assessed by LC-MS and are shown in TableIII. Because of these results, HEK293 is chosen as host cell line forimproved rFIX isoform screening.

A broad panel of HEK293-derived rFIX producing cell lines is generatedand screened for rFIX productivity and clotting activity by theestablished techniques. rFIX secreted from these cell clones is preparedand analyzed according to the above outlined procedures, and exhibits atleast 25% phosphorylated and sulfated isoform of total rFIX. Someexamples of HEK293-derived cell clones and their characteristic rFIXcell specific productivity rates determined by ELISA and APTT are listedin Table IV. Also listed are the percentages determined by LC-MS offully phosphorylated and sulfated FIX isoform found after chromatograhicpurification. As a control, the phosphorylation and sulfation content ofa pdFIX product is determined by the same MS analytical procedure. Thevalues of the CHO derived rFIX product Benefix™ taken from Kaufman etal., 1986, and Wasley et al., 1993, are also given in Table IV.

TABLE III Comparison of percentages of phosphorylated and sulfated rFIXisoforms equally expressed in 4 different host cell types, purified fromcell culture supernatants and determined by LC-MS. Percentage ofsulfated and Percentage of sulfated and phosphorylated rFIXphosphorylated rFIX isoform after enrichment isoform after usualaccording to the present Host Cell type purification invention CHO 2-4%not determined BHK  2% not determined SkHep not detected not determinedHEK293 10-20%  25-56%

TABLE IV Cell specific production rates per day based on ELISA and APTTdata, and percentages of fully phosphorylated and sulfated rFIX isoformproduced by seven examples of HEK293-derived cell lines. The percentageof phosphorylated and sulfated FIX isoform found in pdFIX, andcorresponding values of rFIX producing CHO clones taken from literatureare also given. Percentage of fully phosphorylated and μg FIX/ mU FIX/sulfated FIX 10⁶ cells/ 10⁶ cells/ material after day day purificationHEK293 clone #1 3.2 50 25% HEK293 clone #2 1.6 130 28% HEK293 clone #32.6 360 35% HEK293 clone #4 10 1200 53% HEK293 clone #5 25 2900 46%HEK293 clone #6 26 3300 49% HEK293 clone #7 19 1300 53% HEK293 clone #84.6 570 56% pdFIX — — 98% CHO-derived cell clones; 2-4 90-300 <1% valuestaken from literature

Example 3 Pharmacokinetics of Improved, HEK293-Derived rFIX inComparison with pdFIX and rFIX Product Benefix™

The goal of the study is to confirm a significantly improved in vivorecovery but same half-life of HEK293cell-derived rFIX in comparison toCHO cell-derived Benefix™ when administered to FIX-knockout mice. Inaddition, a pdFIX preparation should reveal similar pharmacokineticproperties as the HEK293-derived protein and serve as a control to showvalidity of the chosen animal model and to comply with literature data.

HEK293 cell lines producing high-phosphorylated and sulfated rFIX can beused for production of the test substance. rFIX can be purified fromcell culture supernatants by semi-affinity calcium-dependent anionexchange chromatography (EP 0669342). Benefix™ and a pdFIX product areboth commercially available. Degrees of phosphorylation and sulfationcan be analyzed by LC-MS.

Briefly, rFIX and pdFIX forms can be administered i.v. in FIX-knockoutmice at a dosage of 250 μg/kg and 10 ml/kg. Activity and concentrationof administered FIX can be determined in plasma samples taken atmultiple time points by ELISA and APTT clotting assay to calculatepharmacokinetic parameter values.

As shown in Example 2, several rFIX expressing HEK293 cell lines havebeen screened for performing high degrees of rFIX phosphorylation andsulfation. Frozen cells are stored in liquid nitrogen and cells can besuspended in DMEM/Ham's F12 medium containing 5-10% fetal bovine serumto larger culture systems like triple-T flasks. At confluency, the cellsshould be switched to vitamin K1-containing serum-free medium.Supernatants can be harvested every day for up to two weeks. rFIX can bepurified via semi-affinity calcium-dependent anion exchangechromatography in an endotoxin-free system. Final product should beformulated in Benefix™ formulation buffer (10 mM L-histidine, 260 mMglycine, 1% sucrose, 0.005% Tween-80 in water, pH 6.8) at aconcentration of 250 μg/10 ml and analyzed by LC-MS for degrees ofphosphorylation and sulfation.

Benefix™ can be reconstituted in formulation buffer (10 mM L-histidine,260 mM glycine, 1% sucrose, 0.005% Tween-80, pH 6.8) at a concentrationof 250 μg/10 ml. The pdFIX can be reconstituted in Aqua bidest.,dialyzed against Benefix™ formulation buffer and adjusted in this bufferto a concentration of 250 μg/10 ml. The pdFIX and Benefix™ should beanalyzed by LC-MS for degrees of phosphorylation and sulfation.

A single dose of FIX preparations at 250 μg/kg and 10 ml/kg in Benefix™formulation buffer can be administered via the lateral tail vein ofFIX-knockout mice. Per time point and treatment, 5 male and 5 femaleanimals should be used. Mice must be anesthetized, and blood can becollected by cardiac puncture at 15 min, 30 min, 1 hr, 4 hrs, 9 hrs postinjection into sodium citrate to a final ratio of 1:10 (citrate:blood)and a final sodium citrate concentration of 3.8%. Plasma samples shouldbe frozen immediately after centrifugation.

A control group of 5 male and 5 female mice administered with bufferonly can be done at the first and the final time point.

All plasma samples can be tested for rFIX or pdFIX concentrations andactivities by using ELISA and APTT clotting assay against FIX standards.Aliquots of the FIX preparations for injection should be used asreference substances to determine the actual injected amounts.

At least, the following pharmacokinetic parameters should be calculatedto estimate improved pharmacokinetic properties:

-   -   a. In vivo recovery: time point with highest concentration of        ELISA or APTT values compared to injected dose (U/dL or μg/dL        increase per U/kg or μg/kg injected).    -   b. Elimination half-life (one-phase least square linear        regression model of logarithmic transformed ELISA and APTT        values).

Example 4 Transient Transfection and Co-Expression of rVKORC1 inrFIX-Producing HEK293- and CHO-Derived Cell Lines

The expression of rFIX is achieved by introducing expression plasmidscontaining the human FIX encoding DNA sequence under the control of astrong viral promoter into mammalian host cell lines by an appropriatetransfection method resulting in cells having the introduced sequencesstably integrated into their genomes. The plasmids also conferresistance to a selectable marker drug by delivering the adequateresistance gene(s). In the case of CHO cells, which are able to growonly in presence of nucleotide precursors in the medium because of anenzyme defect of the nucleotide de-novo synthesis pathway, theexpression of this enzyme, dihydrofolate-reductase (DHFR), is required.This enables co-amplification of the FIX gene by gradually increasingthe concentration of methotrexate (MTX), which leads to an increase ofcopy numbers of both genes, encoding DHFR and rFIX, within the cell'sgenome. For that purpose, CHO derived cell clones have to be grown alsoin selective medium lacking nucleotides and nucleotide precursors.

For the identification of human rFIX producing cells, after transfectionand addition of the selective drug(s) to the medium, the cell suspensionis diluted to enable isolation of single-cell derived clones. Afterisolation, these cell clones are cultivated to confluency to enablemeasurement of rFIX content of the cell culture supernatant by ELISAtechnique. For that purpose, the cells have to be grown in the absenceof any growth promoting fetal bovine serum or components thereof toensure identification of by the cells secreted rFIX. To ensure a fullyfunctional rFIX protein, vitamin K is added. The supernatant isharvested after 24 hours and analyzed by rFIX-specific ELISA technique.In addition, the protein's integrity and activity is assessed bymeasuring APTT.

Co-expression of rVKORC1 is accomplished by transient expressiontechniques using cell lines, which are already selected for rFIXexpression. An expression plasmid comprising rVKORC1 cDNA is transfectedinto these cells without further clone selection. The supernatants arecollected from the whole transfected cell pools, and rFIX content andactivity are compared to negative controls and normalized for specificrFIX secretion rates to assess effects of rVKORC1 activity.

MATERIALS AND METHODS Expression Vectors

The expression vectors are cloned according to standard cloningtechniques. Briefly, pSV-DHFR is generated by inserting the PstI 1.5 kbpfragment of vector pAdD26SV(A)-3 (Scahill et al., 1983; vector is a giftby Dr. Hauser, GBF Germany) containing murine DHFR into a pSVβ vector(Clontech, Palo Alto, Calif.) providing the SV40 enhancer, earlypromoter and intron, where the β-galactosidase gene has been removed byNotI digestion, and a polylinker has been inserted. This vector has alsobeen used to generate phact containing the human actin promoter andintron by exchanging the EcoRI/HindIII fragment with the EcoRI/HindIIIfragment of phβAPr-1-βgal, which is also a gift by Dr. Hauser. phact-FIXcontaining wild-type human FIX cDNA with the ala148 polymorphism (McGrawet al., 1985) is generated by EcoRI digestion of pFIX-bluescript, whichhas been generated by inserting human FIX from a randomly primed humanliver cDNA library into pBluescript (Stratagene, La Jolla, Calif.), andinserting the resulting fragment into phact partially digested withEcoRI.

The vector pCMV-FIX-neo is generated by inserting the EcoRI fragment ofvector pFIX-bluescript into pCMVβ (Clontech), where the β-gal cDNA hasbeen removed. Within this vector, the codon for Ala is exchanged to Thrby site-specific mutagenesis via PCR, changing the naturally occurringpolymorphism of Ala148 to Thr148. The PCR product is re-inserted intothe same vector again. The EcoRI fragment of this vector is cloned intopcDNA3.1 (Invitrogen, Carlsbad, Calif.) to yield pCMV-FIX-neo.

The vector pCMV-VKORC1-EDHpro is generated by using the vectorpCEP4-VKORC1 (kindly provided by Prof. Oldenburg, for description seeRost et al., 2004) as a template for PCR. The PCR product containing therVKORC1 cDNA is cloned into the pCMV-EDHpro vector (Herlitschka et al.,1996).

Cell Culture and Transfections

CHO DUKX/DXB11 cells were obtained from Columbia University (New York,N.Y.) and were cultivated in DMEM/Ham's F12 (1:1) mix (Invitrogen)supplemented with 5% fetal bovine serum (PAA, Linz, Austria),desoxy-adenosine, adenosine and thymidine (all from Sigma, St. Louis,Mo.) and L-Glutamine (Invitrogen) and penicillin/streptomycin(Invitrogen). HEK293 cells (ATCC No. CRL-1573) are cultivated inDMEM/Ham's F12 (1:1) mix supplemented with 5% fetal bovine serum andL-Glutamine and penicillin/streptomycin. For stable transfections, acalcium-phosphate co-precipitation method is used. CHOrFIX cells aregenerated by co-transfection with the linearized plasmids phact-FIX andpSV-DHFR and by selection in DMEM/Ham's F12 (1:1) mix withouthypoxanthine, glycine, and thymidine (Invitrogen) supplemented with 5%dialyzed FBS (PAA). For gene amplification, MTX (Ebewe, Unterach,Austria) is added in stepwise increased concentrations beginning with 10nM up to 200 nM. HEK293 cells are transfected with linearized plasmidpCMV-FIX-neo and selected in medium containing 500 μg/ml G418(Invitrogen). Cell clones are isolated by limited dilution cloningtechniques either manually or using a flow cytometric cell sortingtechnique.

FIX secretion into cell culture supernatants is detected by exchangingthe growth medium for serum-free medium supplemented with 10 μg/mlvitamin K1 (Sigma). Supernatants are collected and. FIX concentrationsare determined by ELISA and clotting assay (APTT). For the calculationof specific secretion rates, cell numbers are counted using a CASY cellcounter (Schärfe Systems, Reutlingen, Germany).

For transient co-expression experiments, the non-linearized plasmidpCMV-VKORC1-EDHPro is transfected using Lipofectamine 2000 reagent(Invitrogen). The same vector without rVKORC1 cDNA is used as negativecontrol.

Analytical Methods

ELISAs are performed using a polyclonal rabbit anti-human FIX (AccurateChemical, Westbury, N.Y.) in a 1:40000 dilution as primary antibody, anda polyclonal goat anti-human FIX horseradish-peroxidase conjugate asdetection antibody. As a standard, a human pd FIX (Enzyme ResearchLaboratories, S. Lafayette, Ind.) is used. APTT is determined using aSTA Compact automated coagulometer (Diagnostica Stago, Asnieres, France)by diluting FIX-samples into FIX deficient plama. All reagents forclotting are from Baxter, Vienna, Austria.

Results

Two stable rFIX-producing cell lines, one CHO- and one HEK293-derived,are subjected to transient transfections with the expression vectorpCMV-VKORC1-EDHpro carrying a cDNA encoding human VKORC1. As controls,the empty vector pCMV-EDHpro and the stable rFIX-expressing cell lineare used. After transient transfections, the cells are left overnight inserum-containing medium. The cells are washed with PBS and cultivatedfor 24 hours in serum-free medium, then the supernatants are harvested.rFIX expression and secretion into the medium is monitored byimmunochemical and coagulation diagnosis methods measuring antigen levelor clotting activity. To estimate effects on cellular productivity, thesecretion rates are calculated on the basis of product concentration percell number and 24 hours.

HEK293 cells expressing rFIX shows a 2.7-fold mean increase of specificsecretion rates and a 2.9-fold increase of rFIX-concentrations afterrVKORC1 transfection in comparison to the empty vector control. Thesevalues are based on APTT measurements. ELISA values shows a 2.0-foldincrease of concentrations, and a 1.8-fold increase of specificproductivities.

For the CHO-derived rFIX-producer cell line, a 1.5-fold increase ofELISA-titers, and a 1.2-fold increase of ELISA-based specific secretionrates are observed. APTT-calculated secretion rates are 1.4-fold higher,and APTT-measured FIX concentrations 1.7-fold.

REFERENCES

Arruda, V. R., Hagstrom, J. N., Deitch, J., Heiman-Patterson, T.,Camire, R. M., Chu, K., Fields, P. A., Herzog, R. W., Couto, L. B.,Larson, P. J., and High, K. A., 2001. Posttranslational modifications ofrecombinant myotube-synthesized human factor IX. Blood 97, 130-138.

Bjorkman, S., Shapiro, A. D., and Berntorp, E., 2001. Pharmacokineticsof recombinant factor IX in relation to age of the patient: implicationsfor dosing in prophylaxis. Haemophilia 7, 133-139.

Bond, M., Jankowski, M., Patel, H., Karnik, S., Strang, A., Xu, B.,Rouse, J., Koza, S., Letwin, B., Steckert, J., Amphlett, G., and Scoble,H., 1998. Biochemical characterization of recombinant factor IX. Semin.Hematol. 35, 11-17.

Brinkhous, K. M., Sigman, J. L., Read, M. S., Stewart, P. F., McCarthy,K. P., Timony, G. A., Leppanen, S. D., Rup, B. J., Keith, J. C., Jr.,Garzone, P. D., and Schaub, R. G., 1996. Recombinant human factor IX:replacement therapy, prophylaxis, and pharmacokinetics in caninehemophilia B. Blood 88, 2603-2610.

Ewenstein, B. M., Joist, J. H., Shapiro, A. D., Hofstra, T. C.,Leissinger, C. A., Seremetis, S. V., Broder, M., Mueller-Velten, G., andSchwartz, B. A., 2002. Pharmacokinetic analysis of plasma-derived andrecombinant F IX concentrates in previously treated patients withmoderate or severe hemophilia B. Transfusion 42, 190-197.

Franck, N., Le Seyec, J., Guguen-Guillouzo, C., and Erdtmann, L., 2005.Hepatitis C virus NS2 protein is phosphorylated by the protein kinaseCK2 and targeted for degradation to the proteasome. J. Virol. 79,2700-2708.

Harrison, S., Adamson, S., Bonam, D., Brodeur, S., Charlebois, T.,Clancy, B., Costigan, R., Drapeau, D., Hamilton, M., Hanley, K., Kelley,B., Knight, A., Leonard, M., McCarthy, M., Oakes, P., Sterl, K.,Switzer, M., Walsh, R., and Foster, W., 1998. The manufacturing processfor recombinant factor IX. Semin. Hematol. 35, 4-10.

Herlitschka, S. E., Falkner, F. G., Schlokat, U., & Dorner, F., 1996.Overexpression of human prothrombin in permanent cell lines using adominant selection/amplification fusion marker. Protein Expr. Purif, 8,358-364.

Kaufman, R. J., 1998. Post-translational modifications required forcoagulation factor secretion and function. Thromb. Haemost. 79,1068-1079.

Kaufman, R. J., Wasley, L. C., Furie, B. C., Furie, B., and Shoemaker,C. B., 1986. Expression, purification, and characterization ofrecombinant gamma-carboxylated factor IX synthesized in Chinese hamsterovary cells. J. Biol. Chem. 261, 9622-9628.

Keith, J. C., Jr., Ferranti, T. J., Misra, B., Frederick, T., Rup, B.,McCarthy, K., Faulkner, R., Bush, L., and Schaub, R. G., 1995.Evaluation of recombinant human factor IX: pharmacokinetic studies inthe rat and the dog. Thromb. Haemost. 73, 101-105.

Kim, Y. M., Barak, L. S., Caron, M. G., and Benovic, J. L., 2002.Regulation of arrestin-3 phosphorylation by casein kinase II. J. Biol.Chem. 277, 16837-16846.

Kisker, C. T., Eisberg, A., and Schwartz, B., 2003. Prophylaxis infactor IX deficiency product and patient variation. Haemophilia 9,279-284.

Larson, P. J. and High, K. A., 2001. Gene therapy for hemophilia B:AAV-mediated transfer of the gene for coagulation factor IX to humanmuscle. Adv. Exp. Med. Biol. 489, 45-57.

Lindsay, M., Gil, G. C., Cadiz, A., Velander, W. H., Zhang, C., and VanCott, K. E., 2004. Purification of recombinant DNA-derived factor IXproduced in transgenic pig milk and fractionation of active and inactivesubpopulations. J. Chromatogr. A 1026, 149-157.

Manno, C. S., 2003. The promise of third-generation recombinant therapyand gene therapy. Semin. Hematol. 40, 23-28.

McCarthy, K., Stewart, P., Sigman, J., Read, M., Keith, J. C., Jr.,Brinkhous, K. M., Nichols, T. C., and Schaub, R. G., 2002.Pharmacokinetics of recombinant factor IX after intravenous andsubcutaneous administration in dogs and cynomolgus monkeys. Thromb.Haemost. 87, 824-830.

McGraw, R. A., Davis, L. M., Noyes, C. M., Lundblad, R. L., Roberts, H.R., Graham, J. B., & Stafford, D. W., 1985 Evidence for a prevalentdimorphism in the activation peptide of human coagulation factor IX.PNAS USA, 82, 2847-2851.

Poon, M. C., Lillicrap, D., Hensman, C., Card, R., and Scully, M. F.,2002. Recombinant factor IX recovery and inhibitor safety: a Canadianpost-licensure surveillance study. Thromb. Haemost. 87, 431-435.

Ragni, M. V., Pasi, K. J., White, G. C., Giangrande, P. L., Courter, S.G., and Tubridy, K. L., 2002. Use of recombinant factor IX in subjectswith haemophilia B undergoing surgery. Haemophilia. 8, 91-97.

Rost S., Fregin A., Ivaskevicius V., Conzelmann E., Hortnagel K., PelzH. J., Lappegard K., Seifried E., Scharrer I., Tuddenham E. G., MullerC. R., Strom T. M., Oldenburg J., 2004. Mutations in VKORC1 causewarfarin resistance and multiple coagulation factor deficiency type 2.Nature February 5; 427(6974):537-41.

Roth, D. A., Kessler, C. M., Pasi, K. J., Rup, B., Courter, S. G., andTubridy, K. L., 2001. Human recombinant factor IX: safety and efficacystudies in hemophilia B patients previously treated with plasma-derivedfactor IX concentrates. Blood 98, 3600-3606.

Scahill, S. J., Devos, R., Van der, H. J., & Fiers, W., 1983. Expressionand characterization of the product of a human immune interferon cDNAgene in Chinese hamster ovary cells. PNAS USA, 80, 4654-4658;

Schaub, R., Garzone, P., Bouchard, P., Rup, B., Keith, J., Brinkhous,K., and Larsen, G., 1998. Preclinical studies of recombinant factor IX.Semin. Hematol. 35, 28-32.

Schwaab, R. and Oldenburg, J., 2001. Gene therapy of hemophilia. Semin.Thromb. Hemost. 27, 417-424.

Shapiro, A. D., Di Paola, J., Cohen, A., Pasi, K. J., Heisel, M. A.,Blanchette, V. S., Abshire, T. C., Hoots, W. K., Lusher, J. M., Negrier,C., Rothschild, C., and Roth, D. A., 2005a. The safety and efficacy ofrecombinant human blood coagulation factor IX in previously untreatedpatients with severe or moderately severe hemophilia B. Blood 105,518-525.

Shapiro, A. D., Korth-Bradley, J., and Poon, M.-C., 2005b. Use ofpharmacokinetics in the coagulation factor treatment of patients withhaemophilia. Haemophilia 11, 571-582.

VandenDriessche, T., Collen, D., and Chuah, M. K., 2001. Viralvector-mediated gene therapy for hemophilia. Curr. Gene Ther. 1,301-315.

Wasley, L. C., Rehemtulla, A., Bristol, J. A., and Kaufman, R. J., 1993.PACE/furin can process the vitamin K-dependent pro-factor IX precursorwithin the secretory pathway. J. Biol. Chem. 268, 8458-8465.

White, G. C., Beebe, A., and Nielsen, B., 1997. Recombinant factor IX;Thromb. Haemost. 78, 261-265.

White, G. C., Pickens, E. M., Liles, D. K., and Roberts, H. R., 1998.Mammalian recombinant coagulation proteins: structure and function.Transfus. Sci. 19, 177-189.

All publications, patent publications, patents, and Genback AccessionNos. applications cited in this specification are herein incorporated byreference in their entirety for all purposes as if each individualpublication, patent publication, or patent were specifically andindividually indicated to be incorporated by reference.

1-11. (canceled)
 12. A method of expressing recombinant bloodcoagulation human factor IX (rFIX), wherein at least 25% of the rFIX isfully phosphorylated and sulfated, the method comprising the step ofexpressing a nucleic acid encoding rFIX into an HEK 293 host cell type,said host cell comprising proteins with the ability to performphosphorylation and sulfation as posttranslational modifications;thereby producing rFIX that is at least 25% fully phosphorylated andsulfated.
 13. The method of claim 12, wherein the rFIX is expressed inthe presence of viramin K.
 14. The method of claim 12, wherein saidnucleic acid encoding rFIX comprises a promoter operably linked to asequence that encodes rFIX
 15. The method of claim 14, wherein saidpromoter is the CMV promoter.
 16. The method of claim 12, furthercomprising the step of expressing a nucleic acid encoding furin into theHEK 293 host cell type.
 17. The method of claim 12, wherein said rFIX isless than 98% fully phosphorylated and sulfated.